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Effects of metoclopramide on isolated guinea-pig colon. 2)Interference with ganglionic stimulant drugs
EUROPEAN JOURNAL OF PHARMACOLOGY 12 (1970) 332-341. NORTH-HOLLAND PUBLISHING COMPANY
EFFECTS
OF METOCLOPRAMIDE
2) I N T E R F E R E N C E
ON ISOLATED
WITH GANGLIONIC
GUINEA-PIG
STIMULANT
COLON.
DRUGS
Clementina BIANCHI, L. BEANI and C. CREMA Department of Pharmacology, University of Pisa, Pisa, ltaly
Received 13 February 1970
Accepted 10 June 1970
Clementina BIANCHI, L. BEANI and C. CREMA, Effects of metoclopramide on isolated guinea-pig colon. 2) Interference with ganglionic stimulant drugs, European J. Pharmaeol. 12 (1970) 332-341. Possible effects of metoclopramide on intramural nervous structures of isolated guinea-pig distal colon were investigated to ascertain if stimulation produced by the drug on the gastrointestinal peristalsis depended on actions other than the demonstrated peripheral sensitization to acetylcholine. Nerve-mediated responses were obtained with nicotine and 5-hydroxytryptamine, which stimulated not only cholinergic neurones, but also non-cholinergic nervous structures in the presence of postganglionic cholinergic and adrenergic blockade. Metoclopramide enhanced the cholinergic responses to nicotine, but blocked the others. Conversely, the drug completely prevented the effects of 5-hydroxytryptamine. Since: (i) the stimulatory effect of nicotine on non-adrenergic, non-cholinergic structures was abolished during 5-hydroxytryptamine tachyphylaxis; (ii) 5-hydroxytryptamine maintained its own effects during nicotine tachyphylaxis; (iii) metoclopramide antagonized 5-hydroxytryptamine, it is suggested that the drug blocks tryptaminergic receptors, necessary for activating unknown nervous elements involved in the control of gastrointestinal tone and motility. Metoclopramide
Gu inea-pig colon
1. INTRODUCTION In the previous paper (Beani et al., 1 9 7 0 ) i t was demonstrated that metoclopramide (M) selectively sensitized the smooth muscle cells o f guinea-pig isolated colon to acetylcholine, without changing acetylcholine release or muscle membrane electrical activity. However, this finding did not explain why morphine, but not hexamethonium, fully counteracted the stimulatory effect o f M on tone, although both drugs are able to reduce acetylcholine release from intestinal preparations (Paton, 1957; Beani et al., 1969). Thus, some kind o f influence by M on intramural inhibitory nervous structures was postulated, in agreement with Jacoby and Brodie (1967). The guinea-pig distal colon was chosen to check this hypothesis, because it exhibits non-cholinergic, nonadrenergic nerve-mediated responses not only to
Nicotine
5-Hydroxytryptamine
transmural stimulation, but also to ganglionic drugs (Bianchi et al., 1968). In this report the effects of M on the responses o f the colon to nicotine and 5-hydroxytryptamine are described. The results confirm the hypothesis that M acts upon nervous intramural mechanisms, possibly controlling both tone and motility.
2. METHODS All the experiments were carried out on guinea-pig isolated distal colon, as previously described (Bianchi et al., 1968). Some preparations were taken from animals submitted to periarterial s y m p a t h e c t o m y a frigore, according to Filogamo and Muti (1968) at the level of the inferior mesentheric artery below the ganglion, 4 - 8 days before experiments.
C.Bianchi et aL, Metoclopramide and ganglionic stimulants The success of sympathectomy was controlled by ascertaining: (i) the ineffectiveness of periarterial stimulation, made below the site of freezing and (ii) the fall in the level of tissue catecholamines on the distal colon. The dosage o~ catecholamines was carried out according to H~iggendal (1963). Freshlyprepared solutions of the following drugs (dissolved in Tyrode solution) were used: metoclopramide HC1, kindly supplied by Lepetit Laboratories, nicotine double tartrate, 5-hydroxytryptamine creatine sulphate, atropine sulphate, reserpine, dissolved as phosphate, propranolol HC1, phenoxybenzamine HC1, bretylium tosilate, tetrodotoxin (Sankyo), hexamethonium bromide, cyproheptadine HC1, eserine sulphate. The final concentrations of the drugs were given as salts, except for reserpine and tetrodotoxin.
3. RESULTS 3.1. Effect o f nicotine on normal colons and after cholinergic and sympathergic blockade Nicotine caused a rapid dose-dependent contrac-
333
tion which was not sustained: the threshold concentration was about 5 × 10-7 g[ml, maximum contraction was achieved with 1 X 10-s g/ml. The response was reproducible at all dose levels, provided that: (i) the time of contact of the drug with the preparation was shorter than three minutes and (ii) three-four washings and 15-20 min intervals were interposed between the doses. If the drug was not washed out the contraction, lasting about two minutes, was followed by a long-lasting relaxation, after which the normal tone resumed and tachyphylaxis developed (Comroe, 1960; Day and Vane, 1963; Kosterlitz and Lee, 1964). In atropine-pretreated (1 × 10-7 to 1 × 10 -6 g/ml) colons, nicotine (5 × 10 -7 to 1 X 10-6 g/ml) was no longer effective (Ambache and Freeman, 1968), but the drug at 2 X 10-6 g/ml or more (up to 5 X 10-s g/ml) gave rise to a biphasic or triphasic response consisting of a rapid, short-lasting inhibition of tone and motility, a large contraction and finally by long-lasting relaxation; the third phase was not always present (Ambache and Edwards, 1951; Burnstock and Holman, 1966) (fig. 1). The irregular occurrence of this last response ('rebound
Fig. 1. The small contraction of guinea-pig distal colon to low dose of nicotine (N, 1 X 10-6 g/ml) is abolished by atropine (A, 1 X 10-7 g/ml), while the high contraction to larger doses (N, 1 X 10-s and 1 X 10-4 g/ml) is changed into a triphasic response. • = Washing. Time scale = 1 rain.
334
C.Bianchi et aL, Metoclopramicle and ganglionic stimulants
inhibition') precluded any attempt to examine its nature by means of pharmacological tools. The analysis, therefore, was focused on the primary inhibition and on the secondary contraction. The degree of the first inhibitory phase was directly related to the tone of preparations and to the drug concentration. In contrast, the second excitatory phase did not seem to be proportional either to the depth of the initial relaxation or to the drug concentration. In some colons with low tone, and consequently showing poor relaxation, the secondary contraction differed from the one observed in the same preparation before adding atropine, only as regards a longer latency and smaller height. To exclude the sympathetic nature of the first inhibitory phase, nicotine was tested in atropine-pretreated colons in the following conditions:
1) in the presence of bretylium 1 to 2 X 10-s g/ml, able to block the relaxation due to electrical stimulation of periarterial sympathetic nerves (Bianchi et al., 1968); 2) in preparations taken from reserpinepretreated animals (5 mg/kg, i.p., 18 hr before killing). In some instances, remaining sympathetic relaxation was removed by adding low doses of both tx- and /3-adrenergic blocking agents (1 × 10 -6 g/ml phenoxybenzamine and 1 to 5 X 10-7 g/ml propranolol); 3) in preparations taken from animals submitted to periarterial sympathectomy a frigore (Filogamo and Muti, 1968) and pretreated, or not, with or- and /3-adrenergic blocking agents as in 2). Nicotine was added, only after ascertaining that the electrical stimulation of the inferior mesentheric artery was ineffective. Moreover, at the end of the experiment, some tissues were examined for their catecholamine contents. In
Fig. 2. A - B : The relaxation to sympathetic stimulation at 20/see for 30 see ($S, panel A) was aboli~l:ed in the presence of bretylium (1 × 10- s g/ml) and atropine (1 × 10 -6 g/ml, panel B); contraction to nicotine, (1 × 10- s g/nL., panel A) was changed to a diphasic response (panel B). C - D : Remaining sympathetic relaxation (SS, panel C) in a colon from a reserpine-pretreated guinea-pig was abolished in the presence of propranolol (1 X 10-7 g/ml) and phenoxybenzamine (1 X 10-6 g/ml, panel D); contraction to nicotine (N, 1 X l 0 -s g/ml, panel C) was changed to a diphasic response (panel D). E - F : Absence of sympathetic relaxation, SS, in a colon of a guinea-pig submitted to pexiaxterial sympathectomy four days before (panel E). After adding propranolol (1 X 10- 7 g/ml), phenoxybenzamine (1 X 10- 6 g/ml) and atropine ( l X l 0 -6 g/ml), the usual diphasic response to nicotine (1 X l 0 -s g/ml) is observed. Washing of nicotine was performed 1 min after adding the drug in panel A, C, E and 3 rain after, in panel B, D and F. Time scale = 1 min.
335
C.Bianchi et ai., Metoclopramide and ganglionic stimulants
eight normal colons the noradrenaline contents were 494 + 35 ng/g, S.E. while in five denervated ones it was 45-+ 9 ng/g; in three others the noradrenaline contents were below the levels of sensitivity of the method (10 ng). In all the three above conditions, nicotine still produced initial inhibition of tone and motility, not substantially different from that observed in normal colons pretreated with atropine only. The secondary contraction was also present (fig. 2).. The indirect evidence, therefore, suggeststhat most of, if not all, the first inhibitory phase is not adrenergic in nature. Nevertheless, as shown later, it is nerve-mediated, possibly through non-adrenergic inhibitory nervous elements present in the intestinal wall (Bennett et al., 1966; Burnstock et al., 1966; Day and Warren, 1967). As regards the nature of the secondary contraction (also seen after transmural stimulation in the presence of atropine, of. Bianchi et al., 1968) the opinions of different workers are conflicting. This response is accounted for by: (i) rebound-excitation (Bennett, 1966); (ii) stimulation of non-eholinergic excitatory neurons (Ambache and Freeman, 1968); and (iii) atropine-insensitive cholinergic contraction (Day and
Warren, 1968). Our observations that the secondary contraction is not related to the depth of the initial relaxation agree more closely with the suggestion advanced by Ambache and Freeman (1968), than with that supported by Bennett (1966), although it is difficult, as yet, to directly prove or disprove the former or the latter. The third hypothesis was tested in some atropinetreated (1 X 10-6 g/nil) colons to which nicotine was given, either alone or together with eserine 1 X l0 -7 g/ml: the esterase inhibitor did not potentiate, and at times slightly reduced, the secondary contraction due to nicotine 5X 10-6 to I X 10-sg/ml (fig. 3), at variance with the finding of Day and Warren (1968) in the rabbit and kitten intestine. Thus, the cholinergic nature of the secondary contraction seems questionable, at least in our preparation. Any role of histamine release in this response was ruled out by repeating the experiment in the presence of cyproheptadine 1 X 10-7 g/ml, which was wholly ineffective (fig. 3). On the other hand, the neurogenic nature of both relaxation and contraction was substantiated by the fact that hexamethonium 5 X 10 -s
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L A-B: Reduction of the response to nicotine (N, 1 X 10-s g/ml) by eserine (1 × 10 - 7 g/ml, added between A and B) in attopine-pretreated colon (1 X 10-6 g/ml, panel A). C-D: Ineffectiveness of cyproheptadine 1 X 10-7 g/ml (added between C and D) on the response to nicotine (N, 1 X 10-s g/ml) of an atropine-pretreated colon (panel C). • = washing. Time scale = 1 min. Fig. 3.
336
C.Bianchi et al., Metoclopramide and ganglionic stimulants
g/ml and t e t r o d o t o x i n 1 t o 5 × 10 -7 g/ml completely prevented the effects of'nicotine.
3.2. Effect o f metoclopramide on the response o f the colon to nicotine As expected, high M concentrations (1 X 10 -4 g/ ml) antagonized nicotine (Hukuhara et al., 1966) owing to blockade o f intramural nervous structures. At lower concentrations, however, (1 X 10 -6 to 1 X 10 -s g/ml), M significantly increased the contractions elicited by low nicotine doses (fig. 4). In six colons, the normal responses to nicotine 2 × 10-6 g/ml to 1 × 10 -s g/ml were, respectively, 48 -+ 6.6 mm, S.E. and 72 -+ 5.4 mm; in the presence o f M 3 X 10-4 g/ml, the responses became 58 -+ 8.3 m m and 76 -+ 4.5 nun. The increase was statistically significant only for the lower nicotine concentration (p < 0.01, Student's t test for paired data). This effect is reminiscent o f the potentiation shown by M on the contraction elicited
by pelvic nerve stimulation at low frequencies, as described in a preceding report (Beani et al., 1970). In contrast with the above finding was the evident antagonism exerted by M against the biphasic responses to nicotine in colons submitted to cholinergic blockade (see above paragraph). As shown in fig. 4, M 3 X 10-6 g/ml strongly reduced and, at 1 X 10 -s g/ml, abolished any response to nicotine 5 X 10 -6 to 5 X 10 - s g/rnl; at times only a small relaxation remained. 3.3. Effect o f 5-hydroxytryptamine nerve-mediated responses o f the colon In a previous paper (Bianchi et al., 1968), it was shown that 5-hydroxytryptamine was devoid o f any direct effect on the muscle cells o f guinea-pig distal colon. Low 5-hydroxytryptamine concentrations (1 × 10 -~ g/ml) gave rise to a contraction which was abol-
Fig. 4. A - B - C : In the guinea-pig colon, the contraction by nicotine (N, 5 × 10-6 g/ml, panel A) is enhanced in the presence of metoclopramide (3 X 10-6 g/ml, panel B, and 1 X 10-s g/ml, panel C). Nicotine was washed out after 90 sec. D - E - F : In an atropine (1 × 10-6g/ml) pretreated colon, the polyphasic response to nicotine (5 × 10-6g/ml, panelD) is abolished in the presence of metoclopramide (1 X 10-s g/ml, panel E) and it recovers only after repeated washings (panel F). Nicotine was washed out after 3 min. Time scale = 1 rain.
C.Bianchiet aL, Metoclopmmide and ganglionic stimulants
ished by atropine. In atropine-pretreated colons, higher 5-hydroxytryptamine concentrations (1 X 10-s to 1 × 10-4 g/ml) caused a biphasic or triphasic response similar to that given by nicotine; it was abolished by tetrodotoxin but not by hexamethonium. These findings were confirmed and extended to other experimental conditions. The outline of the response was the same (i.e. relaxation followed by contraction and sometimes by a third, long-lasting inhibition), even when 5-hydroxytryptamine was tested in atropine-treated colons taken from animals submitted to periarterial sympathectomy or pretreated with reserpine (5 mg/kg, i.p., 18 hr before killing), in the presence or not of a- and fl-adrenergic blocking
Fig. 5. A: The contraction by 5-hydroxytryptamine (H, 5 X 10-7 g/ml) is absent in the presence of metoclopramide (M, 3 X 10-6 g/ml) and partly recovers only after repeated washings (A); B: In atropine (1 X 10-6g/ml) pretreated, sympathetically denervated colon, the polyphasic response to 5-hydroxytryptamine (H, 1 X 10- s g/ml) is nearly abolished in the presence of metoclopramide (3 X 10-6 g/ml) and recovers after repeated washings (A). Time scale = 1 rain.
337
agents (phenoxybenzamine, 1 X 10-6 g/ml and propranolol 1 to 5 X 10-7 g/ml) (fig. 5). It, therefore, seems conceivable that there is a similar neuronal mechanism for responses produced both by nicotine and 5.hydroxytryptamine. At variance with the potentiation exerted b y M on the contraction due to low nicotine doses (abolished by atropine), M (3 × 10-6 to 1 X 10-s g/rot) abolished the responses to low 5-hydroxytryptamine doses in normal colons. Moreover, the drug suppressed the responses to high 5-hydroxytryptamine doses (1 X I0-s to 5 X lO-s g/ml) in colons previously submitted to cholinergic and sympathergic blockade. In a few instances a small relaxation remained (fig. 5). 3.4. Inhibition of non-adrenergic, non-cholinergic responses to nicotine during 5-hydroxytryptamine tachyphylaxis On the ground of the generally accepted views (Volle, 1966; Kharkevic, 1967), the blockade of nicotine receptors does not affect the neuronal response to 5-hydroxytryptamine. Accordingly, as shown in fig. 6, 5-hydroxytryptamine 1 X 10-s g]ml gave rise to the expected effect, both in normal and atropine-treated colons, during nicotine tachyphylaxis obtained b y repeating two doses of the drug (1 X 10-s g/ml) without washing. When the amine was added to the atropine-treated colons a few minutes after the second dose of nicotine a slight reduction in t h e response to 5-hydroxytryptamine was observed with respect to that shown after washing out, carefully, nicotine (see on the right panel of fig. 6). Likewise, during 5-hydroxytryptamine tachyphylaxis produced by two subsequent 'high doses without washing (fig. 7), the cholinergic nicotinic receptors were still available, as shown by maintained responses of the colon to pelvic nerve stimulation and to nicotine (panel A, fig. 7). In contrast, if the experiments were repeated in atropine-pretreated colons, nicotine was no longer effective at any time of 5-hydroxytryptamine tachyphylaxis and the expected response was resumed only after repeated washings and long-lasting intervals (one hour or more, panel B, fig. 7). An interesting point is that the usual increase in tone produced by M in normal colons was still present during nicotine tachyphylaxis, but was absent during 5-hydroxytryptamine tachyphylaxis.
338
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Fig. 6. Left: During nicotine tachyphylaxis, induced by repeating two doses (N, 1 X 10-5 g/ml) without washing, 5-hydroxytryptamine (H, 1 X 10-5 g/ml) gives rise to the normal response. Right: In the presence of atropine (1 X 10-6 g/ml), H (1 X 10-5 g/ml) is still able to give a triphasic response in the earlier stages of nicotine tachyphylaxis (1 X 10-5 g/ml). After washing N, however, (A), the response to H is larger.
4. DISCUSSION
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The cholinergic n e u r o n e s located in the i n t r a m u r a l plexuses o f guinea-pig distal colon are highly sensitive to ganglionic stimulant drugs as nicotine and 5-hyd r o x y t r y p t a m i n e . These n e u r o n e s seem to be provided with specific receptors for b o t h drugs, because they can be activated b y one of the two ganglionic stimulants during tachyphylaxis to the other. This behaviour reflects the classical view on the existence o f gangliar tryptaminergic receptors, different from
Fig. 7. A: In normal colons, 5-hydroxytryptamine tachyphylaxis, induced by repeating two doses without washing (H, 1 X 10-4 g/ml) was without effect on responses to pelvic nerve stimulation (P, 20/see, for 30 see) and nicotine (N, 1 X 10-s g/ml).
B: In atropine (1 X 10 -6 g/ml) pretreated colon, N (1 X 10-s g/ml) is no longer effective during H tachyphylaxis. The expected response to N partly recovers after washing H (A). Time scale = 1 rain.
339
C.Bianchi et al., Metoclopramide and ganglionic stimulants
nicotinic ones, although some exceptions are described (Daniel, 1968). The scheme, however, does not help to understand the observations made in atropine-treated colons. As described above, the polyphasic response to nicotine was no longer present during 5-hydroxytryptamine tachyphylaxis, while 5-hydroxytryptamine maintained its own effect during nicotine tachyphylaxis obviously due to the ganglion-blocking effect of the high doses of the drug. This fact suggests that the tryptaminergic and nicotinic receptors are arranged 'in series' and not in 'parallel': the tryptaminergic receptors seem to be the second ring of the chain, the first being nicotinic. In other words, nicotine probably releases 5-hydroxytryptamine (Burks and Long, 1967; Thompson et al., 1969; Thompson, 1968), which, in turn, stimulates unknown nervous elements able to relax and, according to Ambache and Freeman (1968), to contract the muscle. The reduction in the response to exogenous 5-hydroxytryptamine observed during the early stage of nicotine tachyphylaxis in atropine-treated colons is well in agreement with this hypothesis: the endogenous, released amine could, in fact, occupy part of the receptors, thus reducing the effectiveness of exogenous 5-hydroxytryptamine added thereafter. The maintenance of an initial inhibition by nicotine in atropine-treated colons taken from animals pretreated with reserpine could argue against the suggestion that the inhibition by nicotine is due to the release of 5-HT. In fact, it is well known that reserpine depletes 5-HT stores. On the other hand, intestinal 5-HT stores show a relative resistance to the depleting effect of the drug (Carlsson, 1966) so that the remaining amount of the amine, released by nicotine may well stimulate, for at least a short time, the inhibitory structures. The guinea-pig distal colon differs from the stomach of the same animal because, in the latter, the non-adrenergic inhibitory nervous structures are not only directly activated by nicotine and 5-hydroxytryptamine, but also by extrinsic cholinergic nerves (Biilbring and Gershon, 1967). In fact, vagal stimulation relaxes the stomach in the presence of atropine, while pelvic nerve stimulation is ineffective (Bianchi et al., 1968). Moreover, the drive of vagal inhibition is partly nicotinic, partly tryptaminergic, both receptors being 'in parallel' (Biilbring and Gershon, 1968). The physiological meaning of this different nerv-
ous arrangement may be that the extrinsic nervous control of motility is less important in the colon than in the stomach. At variance with the hypothetical scheme suggested by B~ilbring and Gershon (1968), the scheme shown in fig. 8 may be suggested for the colon. Obviously, the drawing of a 'tryptaminergic neuron' means a 5-hydroxytryptamine 'storage site' capable of releasing the amine, when nicotine or acetylcholine act upon it. No direct data are available concerning the level and type of nervous activity in the isolated guinea-pig distal colon at rest. As judged by the effects exerted by atropine and tetrodotoxin on the tone and spike frequency (Beani et al., 1970) and by the effect of hexamethonium on acetylcholine release (Beani et al., 1969), a certain degree of nervous cholinergic drive seems to contribute to the overall motor activity and tone of this intestinal preparation. Therefore, a negative feed-back mechanism, controlled by 5-hydroxytryptamine, could also operate in resting conditions. As discussed in a previous report (Beani et al., 1970), the effect of M on the tone of the distal colon cannot entirely be explained by its sensitizing action on smooth muscle muscarinic receptors. The antagon-
" I
=5HT reteprors inic receptors
= ni tot
® =
hyporhetica! receptors
[ E
Fig. 8. A diagram of possible arrangement of the efferent neurones in the intramural plexuses of guinea-pig distal colon. C = cholinergic neurones, part of them driven by pelvic nerve preganglionic fibers; 5-HT = tryptaminergic neurones or 'storage sites', acting upon cholinergic, inhibitory (I) and excitatory (E) nervous elements. Both nicotinic (.) and 5-HT(o) receptors are represented in the cholinergic neurones, whilst only the latter ones are drawn in the unknown elements. Possible interconnections between I and E ( - - - ~ ) are also suggested. For explanation, see text.
340
C.Bianchi et al., Metoclopramide and ganglionic stimulants
ism e x e r t e d by m o r p h i n e , but not by h e x a m e t h o n i u m , suggests that M, s o m e h o w , interferes w i t h the intramural nervous elements controlling the m o t i l i t y , at r e c e p t o r sites which are different f r o m the nicotinic ones. The additional findings, r e p o r t e d above, that M abolished the nerve-mediated effects o f 5-hydroxyt r y p t a m i n e and no longer affected the t o n e o f the c o l o n during 5 - h y d r o x y t r y p t a m i n e tachyphylaxis, indicate that the drug blocks the t r y p t a m i n e r g i c nervous receptors and requires t r y p t a m i n e r g i c 'activity' in o r d e r to give the e x p e c t e d effects on the tone o f the preparation. In conclusion, the influence o f M on the guinea-pig distal c o l o n seems to d e p e n d on, at least, t w o m e c h anisms: 1) sensitization o f peripheral muscarinic receptors, thus increasing the cholinergic influence on the muscle; 2) antagonism against some 'local horm o n e s ' , as 5 - h y d r o x y t r y p t a m i n e , w h i c h m a y activate u n k n o w n nervous elements involved in the control o f motility. The role actually played by these t w o mechanisms in the action o f the drug in the s t o m a c h remains to be ascertained. In fact it is in this part o f the gastrointestinal tract that intramural inhibitory nervous structures seem to be brought into action by extrinsic nervous reflexes (Jansson, 1969).
ACKNOWLEDGEMENT This investigation was supported by a grant from Consiglio Nazionale deUe Ricerche - Gruppo di Gastroenterologia, Roma.
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Bennett, M.R., G. Burnstock and M.E. Holman, 1966, Transmission from intramural inhibitory ne,to the smooth muscle of guinea-pig taenia coli, J. Physiol. 182,541. Bennett, M.R., 1966, Rebound excitation of the smooth muscle cells of the guinea-pig taenia coli after stimulation of intramural inhibitory nerves, J. Physiol. 185, 124. Bianchi, C., L.Beani, G.M.Frigo and A. Crema, 1968, Further evidence for the presence of non-adrenergic inhibitory structures in the guinea-pig colon, European J. Pharmacol. 4,51. Biilbring, E. and M.D. Gershon, 1967, 5-hydroxytryptamine participation in the inhibitory innervation of the stomach, J. Physiol. 192, 823. Biilbring, E. and M.D. Gershon, 1968, Serotonin participation in the vagal inhibitory pathway to the stomach, Advanc. Pharmacol. 6 A, 323. Burks, T.F. and J.P. Long, 1967, Release of 5-hydroxytryptamine from isolated dog intestine by nicotine, Brit. J. Pharmacol. 30, 229. Burnstock, G. and M.E. Holman, 1966, Effect of drugs on smooth muscle, Ann. Rev. Pharmacol. 6, 129. Burnstock, G., G. Campbell and M. Rand, 1966, The inhibitory innervation of the taenia of the guinea-pig caecum, J. Physiol. 182,504. Carlsson, A., 1966, Drugs which block the storage of 5-hydroxytryptamine and related amines, in: Handbook of Experimental Pharmacology, 5-Hydroxytryptamine and Related Indolealkylamines (Springer, Berlin) 529. Comroe, J.H., 1960, The pharmacological actions of nicotine, Ann. N.Y. Acad. Sci. 90, 48. Day, M. and J.R. Vane, 1963, An analysis of the direct and indirect actions of drugs on the isolated guinea-pig ileum, Brit. J. Pharmacol. 20, 150. Day, M.D. and P.R. Warren, 1967, Inhibitory responses to transmural stimulation in isolated intestinal preparations, J. Pharm. Pharmacol. 10,408. Day, M.D. and P.R. Warren, 1968, A pharmacological analysis of the response to transmural stimulation in isolated intestinal preparations, Brit. J. Pharmacol. 32,227. Daniel, E.E., 1968, Pharmacology of the gastrointestinal tract, in: Handbook of Physiology, Alimentary Canal, Vol. 4, Motility, p. 2267. Filogamo, G. and R. Muti, 1968, Denervazione estrinseca dell'intestino mediante sonda fredda, Boll. Soc. Ital. Biol. Sper. 44, 1158. HSggendal, J., 1963, An improved method for fluorimetric determination of small amounts of adrenaline and noradrenaline in plasma and tissue, Acta Physiol. Scand. 59, 242. Hobbiger, F., F. Mitchelson and M.J. Rand, 1969, The action of some cholinomimetic drugs on the isolated taenia of guinea-pig caecum, Brit. J. Pharmacol. 35, 53. Hukuhara, T., S. Nakayama, H. Fukuda and T. Neya, 1966, Mechanism of metoclopramide on the intestinal movements, Japan. J. Smooth Muscle Res. 2, 22. Jacoby, H.I. and D.A. Brodie, 1967, Gastrointestinal actions of metoclopramide, Gastroenterology 52, 676.
C.Bianchi et al., Metoclopramide and ganglionic stimulants Jansson, G., 1969, Vago-vagal reflex relaxation of the stomach in the cat, Acta Physiol. Scand. 75,245. Ka~ic', T. and V.M. Varagi~, 1968, Effect of increased intraluminal pressure on the release of acetylcholine from isolated guinea-pig ileum, Brit. J. Pharmacol. 32, 185. Kharkevic, D.A., 1967, Ganglionic-Blocking and GanglionicStimulating Agents (Pergamon Press, London). Kosterlitz, H.W. and G.M. Lees, 1964, Pharmacological analysis of intrinsic intestinal reflexes, Pharmacol. Rev. 16, 301. Paton, W.D.M., 1957, The action of morphine and related substances on contraction and on acetylcholine output of coaxially stimulated guinea-pig ileum, Brit. J. Pharmacol. 12, 119.
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Thompson, J.H., 1968, The effect of nicotine on intestinal serotonin levels, European J. Pharmacol. 2, 329. Thompson, J.H., C.A. Spezia and M. Angulo, 1968, Serotonin response to nicotine in immunosympathectomized mice, European J. Pharmacol. 5,391. Volle, R.L., 1966, Muscaxinic and nicotinic stimulant actions at autonomic ganglia, International Encyclopedia of Pharmacology and Therapeutics, Section 12, Vol. 1 (Pergamon Press, London) p. 84. Weisbrodt, N., C.C. Hug, S.K. Schmiege and P. Bass, 1969, Effect of nicotine on guinea-pig taenia coli, Pharmacologist 11,286.
EFFECTS
OF METOCLOPRAMIDE
2) I N T E R F E R E N C E
ON ISOLATED
WITH GANGLIONIC
GUINEA-PIG
STIMULANT
COLON.
DRUGS
Clementina BIANCHI, L. BEANI and C. CREMA Department of Pharmacology, University of Pisa, Pisa, ltaly
Received 13 February 1970
Accepted 10 June 1970
Clementina BIANCHI, L. BEANI and C. CREMA, Effects of metoclopramide on isolated guinea-pig colon. 2) Interference with ganglionic stimulant drugs, European J. Pharmaeol. 12 (1970) 332-341. Possible effects of metoclopramide on intramural nervous structures of isolated guinea-pig distal colon were investigated to ascertain if stimulation produced by the drug on the gastrointestinal peristalsis depended on actions other than the demonstrated peripheral sensitization to acetylcholine. Nerve-mediated responses were obtained with nicotine and 5-hydroxytryptamine, which stimulated not only cholinergic neurones, but also non-cholinergic nervous structures in the presence of postganglionic cholinergic and adrenergic blockade. Metoclopramide enhanced the cholinergic responses to nicotine, but blocked the others. Conversely, the drug completely prevented the effects of 5-hydroxytryptamine. Since: (i) the stimulatory effect of nicotine on non-adrenergic, non-cholinergic structures was abolished during 5-hydroxytryptamine tachyphylaxis; (ii) 5-hydroxytryptamine maintained its own effects during nicotine tachyphylaxis; (iii) metoclopramide antagonized 5-hydroxytryptamine, it is suggested that the drug blocks tryptaminergic receptors, necessary for activating unknown nervous elements involved in the control of gastrointestinal tone and motility. Metoclopramide
Gu inea-pig colon
1. INTRODUCTION In the previous paper (Beani et al., 1 9 7 0 ) i t was demonstrated that metoclopramide (M) selectively sensitized the smooth muscle cells o f guinea-pig isolated colon to acetylcholine, without changing acetylcholine release or muscle membrane electrical activity. However, this finding did not explain why morphine, but not hexamethonium, fully counteracted the stimulatory effect o f M on tone, although both drugs are able to reduce acetylcholine release from intestinal preparations (Paton, 1957; Beani et al., 1969). Thus, some kind o f influence by M on intramural inhibitory nervous structures was postulated, in agreement with Jacoby and Brodie (1967). The guinea-pig distal colon was chosen to check this hypothesis, because it exhibits non-cholinergic, nonadrenergic nerve-mediated responses not only to
Nicotine
5-Hydroxytryptamine
transmural stimulation, but also to ganglionic drugs (Bianchi et al., 1968). In this report the effects of M on the responses o f the colon to nicotine and 5-hydroxytryptamine are described. The results confirm the hypothesis that M acts upon nervous intramural mechanisms, possibly controlling both tone and motility.
2. METHODS All the experiments were carried out on guinea-pig isolated distal colon, as previously described (Bianchi et al., 1968). Some preparations were taken from animals submitted to periarterial s y m p a t h e c t o m y a frigore, according to Filogamo and Muti (1968) at the level of the inferior mesentheric artery below the ganglion, 4 - 8 days before experiments.
C.Bianchi et aL, Metoclopramide and ganglionic stimulants The success of sympathectomy was controlled by ascertaining: (i) the ineffectiveness of periarterial stimulation, made below the site of freezing and (ii) the fall in the level of tissue catecholamines on the distal colon. The dosage o~ catecholamines was carried out according to H~iggendal (1963). Freshlyprepared solutions of the following drugs (dissolved in Tyrode solution) were used: metoclopramide HC1, kindly supplied by Lepetit Laboratories, nicotine double tartrate, 5-hydroxytryptamine creatine sulphate, atropine sulphate, reserpine, dissolved as phosphate, propranolol HC1, phenoxybenzamine HC1, bretylium tosilate, tetrodotoxin (Sankyo), hexamethonium bromide, cyproheptadine HC1, eserine sulphate. The final concentrations of the drugs were given as salts, except for reserpine and tetrodotoxin.
3. RESULTS 3.1. Effect o f nicotine on normal colons and after cholinergic and sympathergic blockade Nicotine caused a rapid dose-dependent contrac-
333
tion which was not sustained: the threshold concentration was about 5 × 10-7 g[ml, maximum contraction was achieved with 1 X 10-s g/ml. The response was reproducible at all dose levels, provided that: (i) the time of contact of the drug with the preparation was shorter than three minutes and (ii) three-four washings and 15-20 min intervals were interposed between the doses. If the drug was not washed out the contraction, lasting about two minutes, was followed by a long-lasting relaxation, after which the normal tone resumed and tachyphylaxis developed (Comroe, 1960; Day and Vane, 1963; Kosterlitz and Lee, 1964). In atropine-pretreated (1 × 10-7 to 1 × 10 -6 g/ml) colons, nicotine (5 × 10 -7 to 1 X 10-6 g/ml) was no longer effective (Ambache and Freeman, 1968), but the drug at 2 X 10-6 g/ml or more (up to 5 X 10-s g/ml) gave rise to a biphasic or triphasic response consisting of a rapid, short-lasting inhibition of tone and motility, a large contraction and finally by long-lasting relaxation; the third phase was not always present (Ambache and Edwards, 1951; Burnstock and Holman, 1966) (fig. 1). The irregular occurrence of this last response ('rebound
Fig. 1. The small contraction of guinea-pig distal colon to low dose of nicotine (N, 1 X 10-6 g/ml) is abolished by atropine (A, 1 X 10-7 g/ml), while the high contraction to larger doses (N, 1 X 10-s and 1 X 10-4 g/ml) is changed into a triphasic response. • = Washing. Time scale = 1 rain.
334
C.Bianchi et aL, Metoclopramicle and ganglionic stimulants
inhibition') precluded any attempt to examine its nature by means of pharmacological tools. The analysis, therefore, was focused on the primary inhibition and on the secondary contraction. The degree of the first inhibitory phase was directly related to the tone of preparations and to the drug concentration. In contrast, the second excitatory phase did not seem to be proportional either to the depth of the initial relaxation or to the drug concentration. In some colons with low tone, and consequently showing poor relaxation, the secondary contraction differed from the one observed in the same preparation before adding atropine, only as regards a longer latency and smaller height. To exclude the sympathetic nature of the first inhibitory phase, nicotine was tested in atropine-pretreated colons in the following conditions:
1) in the presence of bretylium 1 to 2 X 10-s g/ml, able to block the relaxation due to electrical stimulation of periarterial sympathetic nerves (Bianchi et al., 1968); 2) in preparations taken from reserpinepretreated animals (5 mg/kg, i.p., 18 hr before killing). In some instances, remaining sympathetic relaxation was removed by adding low doses of both tx- and /3-adrenergic blocking agents (1 × 10 -6 g/ml phenoxybenzamine and 1 to 5 X 10-7 g/ml propranolol); 3) in preparations taken from animals submitted to periarterial sympathectomy a frigore (Filogamo and Muti, 1968) and pretreated, or not, with or- and /3-adrenergic blocking agents as in 2). Nicotine was added, only after ascertaining that the electrical stimulation of the inferior mesentheric artery was ineffective. Moreover, at the end of the experiment, some tissues were examined for their catecholamine contents. In
Fig. 2. A - B : The relaxation to sympathetic stimulation at 20/see for 30 see ($S, panel A) was aboli~l:ed in the presence of bretylium (1 × 10- s g/ml) and atropine (1 × 10 -6 g/ml, panel B); contraction to nicotine, (1 × 10- s g/nL., panel A) was changed to a diphasic response (panel B). C - D : Remaining sympathetic relaxation (SS, panel C) in a colon from a reserpine-pretreated guinea-pig was abolished in the presence of propranolol (1 X 10-7 g/ml) and phenoxybenzamine (1 X 10-6 g/ml, panel D); contraction to nicotine (N, 1 X l 0 -s g/ml, panel C) was changed to a diphasic response (panel D). E - F : Absence of sympathetic relaxation, SS, in a colon of a guinea-pig submitted to pexiaxterial sympathectomy four days before (panel E). After adding propranolol (1 X 10- 7 g/ml), phenoxybenzamine (1 X 10- 6 g/ml) and atropine ( l X l 0 -6 g/ml), the usual diphasic response to nicotine (1 X l 0 -s g/ml) is observed. Washing of nicotine was performed 1 min after adding the drug in panel A, C, E and 3 rain after, in panel B, D and F. Time scale = 1 min.
335
C.Bianchi et ai., Metoclopramide and ganglionic stimulants
eight normal colons the noradrenaline contents were 494 + 35 ng/g, S.E. while in five denervated ones it was 45-+ 9 ng/g; in three others the noradrenaline contents were below the levels of sensitivity of the method (10 ng). In all the three above conditions, nicotine still produced initial inhibition of tone and motility, not substantially different from that observed in normal colons pretreated with atropine only. The secondary contraction was also present (fig. 2).. The indirect evidence, therefore, suggeststhat most of, if not all, the first inhibitory phase is not adrenergic in nature. Nevertheless, as shown later, it is nerve-mediated, possibly through non-adrenergic inhibitory nervous elements present in the intestinal wall (Bennett et al., 1966; Burnstock et al., 1966; Day and Warren, 1967). As regards the nature of the secondary contraction (also seen after transmural stimulation in the presence of atropine, of. Bianchi et al., 1968) the opinions of different workers are conflicting. This response is accounted for by: (i) rebound-excitation (Bennett, 1966); (ii) stimulation of non-eholinergic excitatory neurons (Ambache and Freeman, 1968); and (iii) atropine-insensitive cholinergic contraction (Day and
Warren, 1968). Our observations that the secondary contraction is not related to the depth of the initial relaxation agree more closely with the suggestion advanced by Ambache and Freeman (1968), than with that supported by Bennett (1966), although it is difficult, as yet, to directly prove or disprove the former or the latter. The third hypothesis was tested in some atropinetreated (1 X 10-6 g/nil) colons to which nicotine was given, either alone or together with eserine 1 X l0 -7 g/ml: the esterase inhibitor did not potentiate, and at times slightly reduced, the secondary contraction due to nicotine 5X 10-6 to I X 10-sg/ml (fig. 3), at variance with the finding of Day and Warren (1968) in the rabbit and kitten intestine. Thus, the cholinergic nature of the secondary contraction seems questionable, at least in our preparation. Any role of histamine release in this response was ruled out by repeating the experiment in the presence of cyproheptadine 1 X 10-7 g/ml, which was wholly ineffective (fig. 3). On the other hand, the neurogenic nature of both relaxation and contraction was substantiated by the fact that hexamethonium 5 X 10 -s
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L A-B: Reduction of the response to nicotine (N, 1 X 10-s g/ml) by eserine (1 × 10 - 7 g/ml, added between A and B) in attopine-pretreated colon (1 X 10-6 g/ml, panel A). C-D: Ineffectiveness of cyproheptadine 1 X 10-7 g/ml (added between C and D) on the response to nicotine (N, 1 X 10-s g/ml) of an atropine-pretreated colon (panel C). • = washing. Time scale = 1 min. Fig. 3.
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C.Bianchi et al., Metoclopramide and ganglionic stimulants
g/ml and t e t r o d o t o x i n 1 t o 5 × 10 -7 g/ml completely prevented the effects of'nicotine.
3.2. Effect o f metoclopramide on the response o f the colon to nicotine As expected, high M concentrations (1 X 10 -4 g/ ml) antagonized nicotine (Hukuhara et al., 1966) owing to blockade o f intramural nervous structures. At lower concentrations, however, (1 X 10 -6 to 1 X 10 -s g/ml), M significantly increased the contractions elicited by low nicotine doses (fig. 4). In six colons, the normal responses to nicotine 2 × 10-6 g/ml to 1 × 10 -s g/ml were, respectively, 48 -+ 6.6 mm, S.E. and 72 -+ 5.4 mm; in the presence o f M 3 X 10-4 g/ml, the responses became 58 -+ 8.3 m m and 76 -+ 4.5 nun. The increase was statistically significant only for the lower nicotine concentration (p < 0.01, Student's t test for paired data). This effect is reminiscent o f the potentiation shown by M on the contraction elicited
by pelvic nerve stimulation at low frequencies, as described in a preceding report (Beani et al., 1970). In contrast with the above finding was the evident antagonism exerted by M against the biphasic responses to nicotine in colons submitted to cholinergic blockade (see above paragraph). As shown in fig. 4, M 3 X 10-6 g/ml strongly reduced and, at 1 X 10 -s g/ml, abolished any response to nicotine 5 X 10 -6 to 5 X 10 - s g/rnl; at times only a small relaxation remained. 3.3. Effect o f 5-hydroxytryptamine nerve-mediated responses o f the colon In a previous paper (Bianchi et al., 1968), it was shown that 5-hydroxytryptamine was devoid o f any direct effect on the muscle cells o f guinea-pig distal colon. Low 5-hydroxytryptamine concentrations (1 × 10 -~ g/ml) gave rise to a contraction which was abol-
Fig. 4. A - B - C : In the guinea-pig colon, the contraction by nicotine (N, 5 × 10-6 g/ml, panel A) is enhanced in the presence of metoclopramide (3 X 10-6 g/ml, panel B, and 1 X 10-s g/ml, panel C). Nicotine was washed out after 90 sec. D - E - F : In an atropine (1 × 10-6g/ml) pretreated colon, the polyphasic response to nicotine (5 × 10-6g/ml, panelD) is abolished in the presence of metoclopramide (1 X 10-s g/ml, panel E) and it recovers only after repeated washings (panel F). Nicotine was washed out after 3 min. Time scale = 1 rain.
C.Bianchiet aL, Metoclopmmide and ganglionic stimulants
ished by atropine. In atropine-pretreated colons, higher 5-hydroxytryptamine concentrations (1 X 10-s to 1 × 10-4 g/ml) caused a biphasic or triphasic response similar to that given by nicotine; it was abolished by tetrodotoxin but not by hexamethonium. These findings were confirmed and extended to other experimental conditions. The outline of the response was the same (i.e. relaxation followed by contraction and sometimes by a third, long-lasting inhibition), even when 5-hydroxytryptamine was tested in atropine-treated colons taken from animals submitted to periarterial sympathectomy or pretreated with reserpine (5 mg/kg, i.p., 18 hr before killing), in the presence or not of a- and fl-adrenergic blocking
Fig. 5. A: The contraction by 5-hydroxytryptamine (H, 5 X 10-7 g/ml) is absent in the presence of metoclopramide (M, 3 X 10-6 g/ml) and partly recovers only after repeated washings (A); B: In atropine (1 X 10-6g/ml) pretreated, sympathetically denervated colon, the polyphasic response to 5-hydroxytryptamine (H, 1 X 10- s g/ml) is nearly abolished in the presence of metoclopramide (3 X 10-6 g/ml) and recovers after repeated washings (A). Time scale = 1 rain.
337
agents (phenoxybenzamine, 1 X 10-6 g/ml and propranolol 1 to 5 X 10-7 g/ml) (fig. 5). It, therefore, seems conceivable that there is a similar neuronal mechanism for responses produced both by nicotine and 5.hydroxytryptamine. At variance with the potentiation exerted b y M on the contraction due to low nicotine doses (abolished by atropine), M (3 × 10-6 to 1 X 10-s g/rot) abolished the responses to low 5-hydroxytryptamine doses in normal colons. Moreover, the drug suppressed the responses to high 5-hydroxytryptamine doses (1 X I0-s to 5 X lO-s g/ml) in colons previously submitted to cholinergic and sympathergic blockade. In a few instances a small relaxation remained (fig. 5). 3.4. Inhibition of non-adrenergic, non-cholinergic responses to nicotine during 5-hydroxytryptamine tachyphylaxis On the ground of the generally accepted views (Volle, 1966; Kharkevic, 1967), the blockade of nicotine receptors does not affect the neuronal response to 5-hydroxytryptamine. Accordingly, as shown in fig. 6, 5-hydroxytryptamine 1 X 10-s g]ml gave rise to the expected effect, both in normal and atropine-treated colons, during nicotine tachyphylaxis obtained b y repeating two doses of the drug (1 X 10-s g/ml) without washing. When the amine was added to the atropine-treated colons a few minutes after the second dose of nicotine a slight reduction in t h e response to 5-hydroxytryptamine was observed with respect to that shown after washing out, carefully, nicotine (see on the right panel of fig. 6). Likewise, during 5-hydroxytryptamine tachyphylaxis produced by two subsequent 'high doses without washing (fig. 7), the cholinergic nicotinic receptors were still available, as shown by maintained responses of the colon to pelvic nerve stimulation and to nicotine (panel A, fig. 7). In contrast, if the experiments were repeated in atropine-pretreated colons, nicotine was no longer effective at any time of 5-hydroxytryptamine tachyphylaxis and the expected response was resumed only after repeated washings and long-lasting intervals (one hour or more, panel B, fig. 7). An interesting point is that the usual increase in tone produced by M in normal colons was still present during nicotine tachyphylaxis, but was absent during 5-hydroxytryptamine tachyphylaxis.
338
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Fig. 6. Left: During nicotine tachyphylaxis, induced by repeating two doses (N, 1 X 10-5 g/ml) without washing, 5-hydroxytryptamine (H, 1 X 10-5 g/ml) gives rise to the normal response. Right: In the presence of atropine (1 X 10-6 g/ml), H (1 X 10-5 g/ml) is still able to give a triphasic response in the earlier stages of nicotine tachyphylaxis (1 X 10-5 g/ml). After washing N, however, (A), the response to H is larger.
4. DISCUSSION
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The cholinergic n e u r o n e s located in the i n t r a m u r a l plexuses o f guinea-pig distal colon are highly sensitive to ganglionic stimulant drugs as nicotine and 5-hyd r o x y t r y p t a m i n e . These n e u r o n e s seem to be provided with specific receptors for b o t h drugs, because they can be activated b y one of the two ganglionic stimulants during tachyphylaxis to the other. This behaviour reflects the classical view on the existence o f gangliar tryptaminergic receptors, different from
Fig. 7. A: In normal colons, 5-hydroxytryptamine tachyphylaxis, induced by repeating two doses without washing (H, 1 X 10-4 g/ml) was without effect on responses to pelvic nerve stimulation (P, 20/see, for 30 see) and nicotine (N, 1 X 10-s g/ml).
B: In atropine (1 X 10 -6 g/ml) pretreated colon, N (1 X 10-s g/ml) is no longer effective during H tachyphylaxis. The expected response to N partly recovers after washing H (A). Time scale = 1 rain.
339
C.Bianchi et al., Metoclopramide and ganglionic stimulants
nicotinic ones, although some exceptions are described (Daniel, 1968). The scheme, however, does not help to understand the observations made in atropine-treated colons. As described above, the polyphasic response to nicotine was no longer present during 5-hydroxytryptamine tachyphylaxis, while 5-hydroxytryptamine maintained its own effect during nicotine tachyphylaxis obviously due to the ganglion-blocking effect of the high doses of the drug. This fact suggests that the tryptaminergic and nicotinic receptors are arranged 'in series' and not in 'parallel': the tryptaminergic receptors seem to be the second ring of the chain, the first being nicotinic. In other words, nicotine probably releases 5-hydroxytryptamine (Burks and Long, 1967; Thompson et al., 1969; Thompson, 1968), which, in turn, stimulates unknown nervous elements able to relax and, according to Ambache and Freeman (1968), to contract the muscle. The reduction in the response to exogenous 5-hydroxytryptamine observed during the early stage of nicotine tachyphylaxis in atropine-treated colons is well in agreement with this hypothesis: the endogenous, released amine could, in fact, occupy part of the receptors, thus reducing the effectiveness of exogenous 5-hydroxytryptamine added thereafter. The maintenance of an initial inhibition by nicotine in atropine-treated colons taken from animals pretreated with reserpine could argue against the suggestion that the inhibition by nicotine is due to the release of 5-HT. In fact, it is well known that reserpine depletes 5-HT stores. On the other hand, intestinal 5-HT stores show a relative resistance to the depleting effect of the drug (Carlsson, 1966) so that the remaining amount of the amine, released by nicotine may well stimulate, for at least a short time, the inhibitory structures. The guinea-pig distal colon differs from the stomach of the same animal because, in the latter, the non-adrenergic inhibitory nervous structures are not only directly activated by nicotine and 5-hydroxytryptamine, but also by extrinsic cholinergic nerves (Biilbring and Gershon, 1967). In fact, vagal stimulation relaxes the stomach in the presence of atropine, while pelvic nerve stimulation is ineffective (Bianchi et al., 1968). Moreover, the drive of vagal inhibition is partly nicotinic, partly tryptaminergic, both receptors being 'in parallel' (Biilbring and Gershon, 1968). The physiological meaning of this different nerv-
ous arrangement may be that the extrinsic nervous control of motility is less important in the colon than in the stomach. At variance with the hypothetical scheme suggested by B~ilbring and Gershon (1968), the scheme shown in fig. 8 may be suggested for the colon. Obviously, the drawing of a 'tryptaminergic neuron' means a 5-hydroxytryptamine 'storage site' capable of releasing the amine, when nicotine or acetylcholine act upon it. No direct data are available concerning the level and type of nervous activity in the isolated guinea-pig distal colon at rest. As judged by the effects exerted by atropine and tetrodotoxin on the tone and spike frequency (Beani et al., 1970) and by the effect of hexamethonium on acetylcholine release (Beani et al., 1969), a certain degree of nervous cholinergic drive seems to contribute to the overall motor activity and tone of this intestinal preparation. Therefore, a negative feed-back mechanism, controlled by 5-hydroxytryptamine, could also operate in resting conditions. As discussed in a previous report (Beani et al., 1970), the effect of M on the tone of the distal colon cannot entirely be explained by its sensitizing action on smooth muscle muscarinic receptors. The antagon-
" I
=5HT reteprors inic receptors
= ni tot
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hyporhetica! receptors
[ E
Fig. 8. A diagram of possible arrangement of the efferent neurones in the intramural plexuses of guinea-pig distal colon. C = cholinergic neurones, part of them driven by pelvic nerve preganglionic fibers; 5-HT = tryptaminergic neurones or 'storage sites', acting upon cholinergic, inhibitory (I) and excitatory (E) nervous elements. Both nicotinic (.) and 5-HT(o) receptors are represented in the cholinergic neurones, whilst only the latter ones are drawn in the unknown elements. Possible interconnections between I and E ( - - - ~ ) are also suggested. For explanation, see text.
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C.Bianchi et al., Metoclopramide and ganglionic stimulants
ism e x e r t e d by m o r p h i n e , but not by h e x a m e t h o n i u m , suggests that M, s o m e h o w , interferes w i t h the intramural nervous elements controlling the m o t i l i t y , at r e c e p t o r sites which are different f r o m the nicotinic ones. The additional findings, r e p o r t e d above, that M abolished the nerve-mediated effects o f 5-hydroxyt r y p t a m i n e and no longer affected the t o n e o f the c o l o n during 5 - h y d r o x y t r y p t a m i n e tachyphylaxis, indicate that the drug blocks the t r y p t a m i n e r g i c nervous receptors and requires t r y p t a m i n e r g i c 'activity' in o r d e r to give the e x p e c t e d effects on the tone o f the preparation. In conclusion, the influence o f M on the guinea-pig distal c o l o n seems to d e p e n d on, at least, t w o m e c h anisms: 1) sensitization o f peripheral muscarinic receptors, thus increasing the cholinergic influence on the muscle; 2) antagonism against some 'local horm o n e s ' , as 5 - h y d r o x y t r y p t a m i n e , w h i c h m a y activate u n k n o w n nervous elements involved in the control o f motility. The role actually played by these t w o mechanisms in the action o f the drug in the s t o m a c h remains to be ascertained. In fact it is in this part o f the gastrointestinal tract that intramural inhibitory nervous structures seem to be brought into action by extrinsic nervous reflexes (Jansson, 1969).
ACKNOWLEDGEMENT This investigation was supported by a grant from Consiglio Nazionale deUe Ricerche - Gruppo di Gastroenterologia, Roma.
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C.Bianchi et al., Metoclopramide and ganglionic stimulants Jansson, G., 1969, Vago-vagal reflex relaxation of the stomach in the cat, Acta Physiol. Scand. 75,245. Ka~ic', T. and V.M. Varagi~, 1968, Effect of increased intraluminal pressure on the release of acetylcholine from isolated guinea-pig ileum, Brit. J. Pharmacol. 32, 185. Kharkevic, D.A., 1967, Ganglionic-Blocking and GanglionicStimulating Agents (Pergamon Press, London). Kosterlitz, H.W. and G.M. Lees, 1964, Pharmacological analysis of intrinsic intestinal reflexes, Pharmacol. Rev. 16, 301. Paton, W.D.M., 1957, The action of morphine and related substances on contraction and on acetylcholine output of coaxially stimulated guinea-pig ileum, Brit. J. Pharmacol. 12, 119.
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Thompson, J.H., 1968, The effect of nicotine on intestinal serotonin levels, European J. Pharmacol. 2, 329. Thompson, J.H., C.A. Spezia and M. Angulo, 1968, Serotonin response to nicotine in immunosympathectomized mice, European J. Pharmacol. 5,391. Volle, R.L., 1966, Muscaxinic and nicotinic stimulant actions at autonomic ganglia, International Encyclopedia of Pharmacology and Therapeutics, Section 12, Vol. 1 (Pergamon Press, London) p. 84. Weisbrodt, N., C.C. Hug, S.K. Schmiege and P. Bass, 1969, Effect of nicotine on guinea-pig taenia coli, Pharmacologist 11,286.